Wenyang Tao, Xingqian Ye, Yanping Cao

a Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048, China

b Zhejiang Key Laboratory for Agro-Food Processing, Fuli Institute of Food Science, Zhejiang R & D Center for Food Technology and Equipment,Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310058, China

Keywords:

β-Carotene

Digestion

Isomerization

cis-β-Carotene

Degradation

ABSTRACT

To investigate the behavior of all-trans-β-carotene during digestion, in-vitro digestion coupled with HPLC-DAD, Raman spectroscopy and Fourier transform-infrared spectroscopy were used to monitor it.All-trans-β-carotene reduced by 75% during the in-vitro digestion and had a highest degradation during intestinal digestion compared with oral and gastro digestion. All-trans-β-carotene occurred isomerization and degradation during oral digestion and occurred degradation during gastro and intestinal digestion.Isomers were identified as 15-cis-β-carotene and 9-cis-β-carotene, degradation products were compounds with function group of C—O, C—O—C or C＝C—C＝C. The biological fate of β-carotene during digestion was clarified, and one of the reasons for low bioavailability of β-carotene was explained by high degradation rate during digestion.

1. Introduction

β-carotene, which is an abundant micronutrient in vegetables and fruits, has been investigated extensively because of its diverse healthrelated functions. As the precursor to vitamin A and as an antioxidant[1], β-carotene is proposed to exhibit a protective effect against cancer[2], heart disease [3], macular degeneration [4], aging [5] and agerelated macular degeneration [6].

However, carotenoids are sensitive to degradation and in particular, to oxidation, because of their single- and double-bond alternation structure. The stability of carotenoids has been a subject of intense research, especially in terms of the stability of β-carotene as a nutritional and bioactive compound. Many researchers have been focused on the stability of β-carotene during food processing and storage. Rodriguez-amaya [7] found that the main critical step for β-carotene loss remains during fruit processing such as juice or puree production. Bechoff et al. [8] found that β-carotene concentration was reduced significantly by the loss of tissue integrity, exposure to light and oxygen and high-temperature thermal processes. Kim et al. [9]investigated the stability of β-carotene in oxygen attack, and showed that β-carotene inside the core of a composite may be protected by cornstarch matrices. Knockaert er al. [10] found that carrot β-carotene occured degradation and isomerization during thermal processing.

However, few investigations have focused on the variation of β-carotene during digestion, the carotene degradation pathway during digestion remains unclear. Digestion has an intensive impact on antioxidants. For example, pH elevation during in-vitro digestion affects the stability of [11,12] polyphenols [11,12], polysaccharides[13,14], vitamins [15] and carotenoids [16,17]. Jayawardena et al. [18]reported that the total polyphenol content changed significantly after digestion, which led to a significant change in antioxidant capacity.Gil-Izquierdo et al. [19] noted that flavonoids, such as hesperidin,were reduced considerably during in-vitro digestion.

The objective of this study was (1) to investigate the stability of β-carotene during in-vitro digestion and (2) to identify the products of isomerization and degradation of β-carotene during in-vitro digestion.The results will help to clarify the biological fate and metabolism pathways of carotenoids during digestion and explain the reasons for the low bioavailability of carotene.

2. Materials and methods

2.1 Chemicals

Porcine pancreatin, bile salt and all-trans-β-carotene were phachased from Sigma-Aldrich (St. Louis, MO, USA). Pepsin and α-amylase were phachased from Shanghai Chemical Reagent Company (China). Decaglyceryl monolaurate (ML750) and medium chain triglycerides (MCT) were phachased from Shanghai Youchuang Co. Ltd (China).

2.2 Emulsion preparation

The pH of 100 mmol/mL NaHCO3solution was adjusted to 7.5 to make buffer solutions. ML750 solutions were stirred overnight to ensure complete dispersion and dissolution.

Emulsions were prepared as Hou et al. [20] described previously with slight modifications. The dispersed phase was prepared by dissolving 1.0 mg β-carotene in 10 mL MCT oil at 37 °C, and stirring at 3 000 r/min overnight in darkness to ensure complete dispersion. The continuous phase was prepared by adding 80 mL 100 mmol/L NaHCO3and 20 mL ML750 into a 250-mL beaker, and stirring for 24 h.

Finally, 125 μL dispersed phase and a different volume (25, 50,75, 100, 125 μL) of continuous phase were combined, and the solution was adjusted to 15 mL and stirred at 1.6 × 105r/min (2.8 × 108g) with a blender to form stable emulsions.

2.3 In-vitro digestion

In-vitro digestion was carried out under the guideness of Minekus et al. [21] and Huang et al. [22] with a few modifications.

Oral digestion: 500 μL of oral digestion juice (60 mg alphaamylase dissolved in 50 mL 1 mmol/L CaCl2solution at pH 7.0) was added and incubated in a shaking water bath for 10 min at 37 °C.

Gastric digestion: the mixture pH was adjusted to 2.0 with 3 mol/L HCl to mimic the acid condition in the gastric. The mixture was incubated for 1 h in a shaking water bath at 37 °C prior to the addition of 0.15 g pepsin .

Intestinal digestion: the mixture was adjusted to pH 6.5 before intestinal juice (0.1 g lipase and 0.625 g bile salts dissolved in 25 mL 0.1 mol/L NaHCO3) were added. Following an adjustment of pH to 7.5 with 1 mol/L NaHCO3, the mixture was incubated for 2 h at 37 °C.

Digested samples: samples went through oral, gastro and intestinal digestion successivly.

2.4 Determination of total carotenoid content

Carotenoid extraction was carried out based on the work by Rodriguez-amaya et al. [23] and Lyan et al. [24]. All samples were extracted with 10 mL hexane at 20 °C for 30 min, and centrifuged for 10 min at 10 000 × g. This progress was repeated 3 times. The supernatant was combined and diluted to 50 mL for further analysis.Absorbances of the diluted samples were measured at 447 nm and quantified with a standard curve.

2.5 Determination of isomers of all-trans-β-carotene

Isomers of all-trans-β-carotene were detected by HPLC-DAD as described by Li et al. [25] with slight modifications. Analyses were performed on an Alliance 2695 separations module (Waters) equipped with a PDA 2996 (Waters) and a YMC carotenoid column (250 × 4.6 mm,5 μm, C30, Japan). Mobile phase A consisted of 82% methanol, 15%methyl tert-butyl ether (MTBE) and 3% water. Mobile phase B consisted of 7% methanol, 90% MTBE and 3% water. The flow rate was 1 mL/min and the column temperature was 25 °C. The gradient elution was performed as follows: 0-100 min, 0%-70% B, the injection volume was 10 μL and the wavelength range recorded was set from 210 nm to 800 nm.

2.6 Determination of degradation products

2.6.1 Fourier transform-infrared spectra (FTIR) detection

FT-IR analysis were carried out as previously described with slight modifications [26]. Samples were lyophilized and blended with KBr powder, ground for 10 min in an agate mortar and then pressed into KBr pellets. FT-IR spectra of samples were collected on Nicolet 5700 spectrometer (Thermo Fisher Scientific, Waltham, USA) using the wavenumber from 4 000 cm-1to 400 cm-1, with resolution of 2 cm-1.

2.6.2 Raman spectra detection

Raman spectra of the degradation products were recorded at 25 °C on a Labor Raman HR-800 (Jobin Yvon Co., France). The covered wave range was 500-2 000 cm-1using the 562 nm excitation line. The power was set to 10 MW. The integration time was 30 s and two spectra were averaged.

2.7 Statistical analysis

All samples were replicated triplicate and the results are presented as mean ± S.D. Regression analyses and other statistical analyses were performed using SPSS Program, version 17.0 (SPSS Inc.,2009). Significant differences between means were determined by Duncan’s multiple range tests. P ＜ 0.05 were chosen as the criteria for statistically significant difference.

3. Results and discussions

3.1 Stability of β-carotene during digestion at different emulsifer concentration

Fig.1 shows the stability ofβ-carotene in the different digestion phases at different emulsifier concentration. It can be found that theβ-carotene was degraded during digestion and had the lowest degradation in gastric phase. The stability ofβ-carotene during gastric phase firstly increased with the increase of emulsifier concentration varied from 25 μL to 75 μL, then decreased with the increase of emulsifier concentration varied from 75 μL to 125 μL, and the stability ofβ-carotene during oral phase had a similar trend. When 25 μL of emulsifier was added, the emulsifier is too low to capture all the oil in the system. Big oil drops were formed with large amounts of free oils which may protect carotene from degradation. As the amount of emulsifier increased to 50 μL, still the emulsifier is too low to capture all the oil in the system. But the amount of free oil was less than 25 μL, which led to smaller oil drops in the system. The bigger the oil drops, the better the protection of carotene. Theβ-carotenoid content were not changed significantly with the increase of emulsion content. The lowest degradation in gastro phase compared with other samples may be explained thatβ-carotene tends to accumulate inside the lipid phase in acidic conditions, which prevent carotenoid from been interacte with amylase and contributes to the protection by the emulsifier [16,17]. The degradation rate (reduced by 75%) (Table 1) ofβ-carotene during digetion was significantly higher than the rate during thermal processing (about reduced by 40%) [27]. The protection of carotenoid during digestion should be emphasized in the future research.

Fig.1 Effects of emulsifier concentration on all-trans-β-carotene stability during digestion. Blank refers to the concentration of blank, which was 0.1 μg/mL. Collums with different letters in the sampe phase are significant different (P ＜ 0.05).

3.2 Identification of all-trans-β-carotene isomers during digestion

HPLC-DAD was used to identify the all-trans-β-carotene and its cis isomers afterin-vitrodigestion. Chromatograms after different stages ofin-vitrodigestion are shown in Fig 2. The isomers ofβ-carotene were identified by comparison with publishedQratios [28-30] (the height ratio of the cis peak (330-350 nm)to the main absorption peak, Table 2) from their respective spectra (Fig. 3). Peak 1 of Fig. 2 was tentatively identified as 15-cis-β-carotene according to theQvalue (Table 1), Peak 3 of Fig. 2 was tentatively identified as 9-cis-β-carotene. The result was different from the result of isomers of thermal processing that the isomerization rates were in the following order: 13-cis-β-carotene ＞15-cis-β-carotene ＞ 9-cis-β-carotene, the conversion from all-transβ-carotene to 13-cis-β-carotene was relatively easy at 25-50°C in oil-in-waterβ-carotene nanoemulsions [31]. 13-cis-β-carotene was the only isomer during drying (60-80 °C) of carrots [32].13-cis-β-carotene was the predominantcis-isomer, whereas 9-cisβ-carotene could only be detected in high temperature sterilized carrot juices during blanching and sterilization [27]. 13-cis-βcarotene was the main isomer, whereas 15-cis- and 9-cis-β-carotene were formed in minor amounts during stir-frying of vegetables[33]. It inferred that isomerization pathway and mechanism was different in digestion and in thermal processing.

Fig. 2 HPLC chromatogram of all-trans-β-carotene in different digestion phases. Peaks: 1, 15-cis-β-carotene; 2, All-trans-β-carotene; 3, 9-cis-β-carotene.

Fig. 3 DAD spectra of the corresponding peaks 1, 2 and 3 of HPLC (Fig. 2).

Table 1Identification of β-carotene cis isomers.

Table 2Peak areas of 15-cis-β-carotene, all-trans-β-carotene and 9-cis-β-carotene in different digestion phases.

3.3 Isomerization and degradation of all-trans-β-carotene during digestion

Theβ-carotene after and before digestion was analysed by HPLC(Fig. 2). It was found that the control sample of all-trans-β-carotene contained minor amounts of two isomers (Peak 1 and Peak 3).During the digesion, the all-trans-β-carotene and its isomers occurred variation. As shown in Fig. 2, all-trans-β-carotene decreased throughout the digestion stages (reduced by 75%). Isomer 1 and 3 increased during oral digestion which was transformed by all-transβ-carotene. For the gastric and intestinal phases, isomer 1 and 3 showed a significant decrease. It can be inferred that all-trans-βcarotene occurred isomerization and degradation during oral phase and occurred degradation during gastric and intestinal phases.

3.4 Degradation products analysis of all-trans-β-carotene during digestion

Fig. 4 showed the FTIR spectra of all-trans-β-carotene during different digestion phases. Three new vibration peaks (2 672, 1 055 and 943 nm) existed afterin-vitrodigestion and one vibration peak at 725 nm faded duringin-vitrodigestion. The new peak at 2 672 nm is ascribed to stretching vibrations of the C—H bonds of aldehyde.The new peak at 1 055 nm indicates that a new polar functional group C—O occurred duringin-vitrodigestion. New peaks at 943 nm indicated that it was reasonable to ascribe the two new peaks to the formation of furanoid, which indicated thatβ-carotene-epoxide orβ-carotene-diepoxide may be formed duringin-vitrodigestion.

Fig. 4 FTIR spectra of all-trans-β-carotene in different digestion phases

Raman spectra are shown in Fig. 5. New peaks were observed in intestinal and digested samples. The occurrence of a peak at 1 660 cm-1represents the formation of four carbon conjugated double bonds (C＝ C—C＝C), which shows that carotene may degraded toβ-ionone andβ-apo-carotenal duringin-vitrodigestion. The Raman signals at about 793 cm-1can be assigned to a C—O—C band, which is part of theβ-carotene-diepoxide and is a kind of furanoid that is formed duringβ-carotene cleavage. The new peak at 606 cm-1that represents the C—COOH band should be ascribed to the hydrolysis reaction of MCT. The new peak at 793 cm-1was present in intestinal samples and disappears in digested samples. The phenomenon may be caused by the synergetic effect of enzymes as Marchese et al. [34] mentioned.

Fig. 5 Raman spectra of all-trans-β-carotene in different digestion phases.

From the occurrence of the new function group (C—O,C—O—C, C＝C—C＝C), all-trans-β-carotene may occur oxidation,cyclization and pyrolysis reaction. In the present study, we only analyzed the function groups of mixed degradation products. The structures of degradation products will be further identified.

4. Conclusion

The behavior ofβ-carotene duringin-vitrodigestion was investigated. The results from this study showed that all-trans-βcarotene occurred isomerization and degradation during oral digestion,and occurred degradation during gastro and intestinal digestion. Two isomers were identified as 15-cis-β-carotene and 9-cis-β-carotene.Degradation products only were analyzed the function groups, new function group (C—O, C—O—C, C＝C—C＝C) appeared. This study gave very useful information on the behavier of all-trans-βcarotene duringin-vitrodigestion and explained the reason of low bioavailabilty from digestion process.

conflict of Interest

The authors declare that they have no conflict of interest.

Acknowledgements

This project was supported by National Natural Science Foundation of China (31771982).